The binding of model drugs to human blood and individual blood components has been determined by equilibrium dialysis and expressed in terms of classes of binding sites, association constants and binding capacities. Chlorpromazine and imipramine are bound to three major components: membranes of red cells, albumin, and lipoproteins. The affinity and capacity of lipoprotein binding is at least as high as that of albumin and is equally distributed on HDL, LDL, VLDL, and on the chylomicrons. White blood cells and platelets are of minor importance in terms of binding capacity. No binding was detected with gamma-globulins or alpha- or beta-globulins other than lipoproteins. In contrast, salicylic acid was not bound to red cells or lipoproteins.

Both UDP-glucuronyltransferase (GT) and β-glucuronidase (ßG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and βG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP; UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn^2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.

Microsomal fraction contains the whole of hepatic UDP-glucuronyltransferase as well as part of β-glucuronidase. The activities of the two enzymes were assayed under identical conditions using untreated male rat liver microsomes at pH 7.5. In a 30-min incubation with p-nitrophenol and UPD-glucuronic acid, a net glucuronide formation of 0.010 μmol-min^-1 •g• -liver^-1 was measured. In the presence of saccharolactone at concentrations selectively blocking β-glucuronidase, the glucuronidation rate was 0.015 μmol•min^-1•g•liver^-1. Using the kinetic parameters of β-glucuronidase (Km = 0.06 mmol/l p-nitrophenylglucuronide, V(m) = 0.075 μmol pNP formed h^-1 •g•liver^-1) determined in the absence of UDP-glucuronic acid, to correct for the β-glucuronidase’s interference on the glucuronidation process, a glucuronide formation of 0.011 μmol•min^-1 •g•liver^-1 was calculated.

6-CB, a model compound which because of its lipophilicity cannot be excreted by the kidneys and is also unmetabolizable, shows an extreme type of pharmacokinetics. In rats given single doses the compound disappears with a half-life of half a life span by fecal excretion. If adipose tissue mass is allowed to increase, the 75% dose initially stored in adipose tissue does not decrease during 280 days. With repeated weekly administration each dose adds some 90% to the body burden. It is concluded that the old hypothesis of drug-metabolizing enzymes as a protective system preventing accumulation of naturally occurring lipophilic drugs is correct.